Steroid receptors are ligand-activated transcription factors which bind to DNA sequences located in the regulatory regions of steroid target genes. Activated receptors can both induce and repress transcription depending on the promoter context and availability of certain transcription factors. Although much is known about the regulation of a few well-studied target genes, less is understood about the determinants of cell-specific steroid regulation. Therefore, based on the accepted model that steroid responses are mediated through changes in the expression of a limited number of target genes, we have been investigating the mechanism of glucocorticoid-induced cell death (apoptosis) in two murine lymphoma cell lines using several different molecular genetic approaches. Our studies of glucocorticoid receptor (GR) structure and function support the notion that steroid regulation of lymphocyte apoptosis is at the level of transcription and have led to preliminary work aimed at the isolation and characterization of lymphocyte target genes. Three independent approaches are described in this proposal which extend our earlier studies and have the following objectives: 1) to use receptor fusions and genetic selections to identify amino acids in the GR amino terminus which are required for steroid-induced cell death, 2) to characterize cDNA sequences which we have recently isolated by subtraction hybridization techniques, and to determine what role they may have in eliciting the cell death phenotype, and 3) to isolate and characterize lymphocyte mutant cell lines which express normal levels of wild-type GR but are steroid-resistant (deathless phenotype). Together these analyses should lead to a better understanding of cell-specific steroid gene networks and potentially identify key target genes required for lymphocyte cell death. Moreover, a genetic selection for steroid-resistance among cells stably transfected with GR variants is significant since it may allow for a more thorough examination of receptor functions such as those requiring protein-protein interactions.